Active inflammatory conditions in the gastrointestinal tract are characterized by migration of neutrophils (PMN) across epithelial mucosal surfaces. Previous studies suggested a central role of CD47 in modulating PMN migration across intestinal epithelium and demonstrated that CD47 on both PMN and epithelial cells acts to enhance PMN transmigration. The goal of this proposal is to further characterize the molecular mechanism(s) by which CD47 regulates PMN transepithelial migration. The specific aims in this proposal are 1) to investigate the role of tyrosine phosphorylation in CD47-mediated PMN transmigration; 2) to determine the subcellular localization of CD47 in PMN and analyze its redistribution after chemoattractant stimulation; 3) to investigate the role of epithelial CD47 in PMN transmigration. In this proposal, CD47- mediated tyrosine phosphorylation in PMN will be analyzed by applying specific tyrosine kinase inhibitors in PMN transmigration assays to examine their ability of reversal anti-CD47 antibody inhibition in transmigration. The specific phospho-protein(s) down stream of CD47 during fMLP stimulation will be identified and characterized by 2D-PAGE, phospho-immunoblotting, and proteomics. Subcellular fractionation by sucrose and percoll gradients will be performed to identify the subcellular localization of intracellular pools of CD47 and compared with the localization of SIRPa (CD47 ligand) and CD11bCD18 in PMN before and after fMLP stimulation. Effect of PI3-kinase inhibition in re-distribution of these proteins will also be investigated. Finally, epithelial cell surface CD47 in PMN migration will be studied using T84 epithelial cells and CD47-transfected CaCO2 cells. The potential functionally important residues (or domains) on CD47 extracellular and intracellular domains will be studied by mutagenesis and expression on CaCO2. Such studies will provide insights into the molecular basis of PMN transepithelial chemotaxis which may lead to new therapeutic inventions in the treatment of acute inflammatory diseases of mucosal surfaces.